Fish of the family Salmonidae are amongst the most commercially important of game fish. This taxonomic family includes such well known species as Atlantic salmon, brown trout, rainbow trout, coho salmon, and arctic char. Several species of salmonid fish are parasitized by the myxozoan parasite Myxobolus spp. (e.g., M. cerebralis, M. insidiosus, M. squamalis) which is responsible for the chronic inflammatory condition called whirling disease. The radical tail chasing behavior of infected fish, which characterizes the disease, results from pressure on the auditory capsule due to developmental stages of the parasite and the accompanying host's inflammatory response. The origins of the pathogen are suspected to be central Europe and Asia but movements of frozen and live trout have spread the agent to a total of 21 countries and 20 of the states in the USA. Recent increases in outbreaks of whirling disease have highlighted the vulnerability of stocks of salmonid fish in state and private hatcheries. The costs associated with control programs can be staggering. From 1965 to the present in California and Michigan alone these costs are estimated to exceed $30,000,000. This has brought to the forefront the concerns of transmission of pathogens between cultured and wild fish populations and the need to readily identify infected fish populations.
The phylum Myxozoa comprises a group of multicellular organisms that principally infect fish (Lom, J., Parasitol. Today 3:327 (1982)). Myxozoans found in freshwater have complex life cycles with alternating forms found in fish and aquatic oligochaete worms (Kent, M. L., et al., Can. J. Zool. 72:932 (1994); Wolf, K., et al., Science 255:1449 (1984); Wolf, K., et al., J. of Fish Dis. 9:83 (1986)). These stages are designated as myxosporean and actinosporean in their respective fish and worm hosts. The parasite stages designated as spores and triactinomyxons, respectively, emerge from the fish and worm hosts and they are infectious only for the other host (Wolf, K., et al., Science 255:1449 (1984)).
There are 6 Myxobolus spp. found in salmonid fish (Amandi, A., et al., Fish Pathol. 20:287 (1985); Hedrick, R. P., et al., J. Aquat. Animal Health 3:55 (1991); Hoffman, G. L., J. Aquat. Animal Health 2:30 (1990); Margolis, L., Aspects of Parasitology, E. Meerovitch, ed., McGill University, Montreal, pp. 135-226 (1982)). It is clear however, that the tropism for, and destruction of, cartilage by M. cerebralis causes it to be the most serious pathogen among this group. Because cartilage is a key structural component of the fish skeleton throughout life and particularly in young fish, it is understandable why the severity of whirling disease is inversely related to the age of the fish (Halliday, M. M., et al., J. of Fish Biol. 9:339 (1976); Markiw, M. E., Aquaculture 91:1 (1991)). It is among these very young trout that mortality has been attributed directly to the pathogen (Markiw, M. E., Aquaculture 91:1 (1991)). And, survivors, which act as carriers, may demonstrate lifelong deformities of the head, jaws, and spinal column because cartilage damage has interfered with subsequent bone deposition.
These pathogenic characteristics combined with the inability to eliminate the pathogen once established in natural waters, has made it the most serious fish pathogen facing the intermountain states of the US. Once believed to be strictly a disease among fish in aquaculture, whirling disease is now believed to be the major cause of the demise of wild rainbow trout populations in Montana, Colorado (Walker, P. G., et al., An investigation to determine the disappearance of young wild rainbow trout in the upper Colorado River in Middle Park, Colo., Colorado Division of Wildlife, Montrose, Colo., 134 pp. (1995)), and Utah. Myxobolus cerebralis is now found in 20 states and appears to be spreading into and devastating wild populations of trout in the intermountain west.
Current methods for identification of M. spp. rely on the observation and measurement of spores as extracted from infected trout. This approach does not detect prespore stages which are present until spores develop 50-60 days post exposure to the infective stage. The problem is further exacerbated by the complex and changing alternate forms of the parasite found in the fish and aquatic oligochaete hosts. Additionally, immunodiagnostic methods have not shown the specificity required to distinguish spores of M. cerebralis from related Myxobolus spp. as found in rainbow trout.
Detection of the causative parasite is currently based on a definitive description of the spore consistent with its characteristic size and shape (Lom J, Hoffman G L (1970) J Parasitol 56: Proc 2nd Int Congr Parasit Abstr No 387) followed by confirmation of developmental stages or spores in cartilage of hematoxylin and eosin (H&E)--stained tissue sections (Thoesen, J C (1994) ed. Suggested Procedures for the Detection and Identification of Certain Fish and Shellfish Pathogens. 4th ed, Version 1, Fish Health Section, American Fisheries Society). The direct fluorescent antibody test has been used on occasion (Markiw M E (1989) J Fish Dis 12:137-141). A combination of digestion (Markiw M E, Wolf K (1974) J Fish Res Bd Can 31:15-20) and centrifugation (O'Grodnick J (1975) J Wildl Dis 11:54-57) techniques has been employed to detect the spores from cartilage in fish with Light infections. Although the later digestion/concentration procedure is effective in detecting the spore stage M. cerebralis, it is time consuming, labor intensive and destroys the prespore stages rendering them undetectable.
Accordingly, what is needed in the art is a means to rapidly detect the presence of M. spp. from an aquatic sample. In particular, what is needed is a means to detect this parasite with high specificity and sensitivity in both the fish and oligochaete hosts. The present invention provides these and other advantages.